Positron emitting radionuclide labeled peptides for human upar pet imaging

ABSTRACT

There is provided a positron-emitting radionuclide labelled peptide for non-invasive PET imaging of the Urokinase-type Plasminogen Activator Receptor (uPAR) in humans. More specifically the invention relates to human uPAR PET imaging of any solid cancer disease for diagnosis, staging, treatment monitoring and especially as an imaging biomarker for predicting prognosis, progression and recurrence.

This application is a divisional of U.S. patent application Ser. No.14/649,113, filed 2 Jun. 2015, which is a National Stage Application ofPCT/DK2013/050402, filed 29 Nov. 2013, which claims benefit of SerialNo. 2012 70751, filed 3 Dec. 2012 in Denmark, and which claims benefitof Ser. No. 61/732,443, filed 3 Dec. 2012 in the United States, andwhich application(s) are incorporated herein by reference. To the extentappropriate, a claim of priority is made to each of the above disclosedapplications.

FIELD OF THE INVENTION

The present invention relates to a positron-emitting radionuclidelabelled peptide for noninvasive PET imaging of the Urokinase-typePlasminogen Activator Receptor (uPAR) in humans. More specifically theinvention relates to human uPAR PET imaging of any solid cancer diseasefor diagnosis, staging, treatment monitoring and especially as animaging biomarker for predicting prognosis, progression and recurrence.

BACKGROUND OF THE INVENTION

Urokinase-type plasminogen activator receptor (uPAR) is over-expressedin a variety of human cancers¹, including prostate cancer (PC), whereuPAR expression in tumor biopsies and shed forms of uPAR in plasma havebeen found to be associated with advanced disease and poor prognosis²⁻⁹.Moreover, in patients with localized PC, high preoperative plasma uPARlevels have been shown to correlate with early progression¹⁰. Consistentwith uPARs important role in cancer pathogenesis, through extracellularmatrix degradation facilitating tumor invasion and metastasis, uPAR isconsidered an attractive target for both therapyl¹¹⁻¹³ and imaging 14and the ability to non-invasively quantify uPAR density in vivo istherefore crucial.

Radiolabeling and in vivo evaluation of a small peptide radiolabeledwith Cu-64¹⁵ and Ga-68¹⁶ have been described for PET imaging of uPAR invarious human xenograft cancer models. Such tracers could specificallydifferentiate between tumors with high and low uPAR expression andfurthermore established a clear correlation between tumor uptake of theuPAR PET probe and the expression of uPAR¹⁵. However, ¹⁸F (t_(1/2)=109.7min; β-F, 99%) is considered the ideal short-lived PET isotope forlabeling of small molecules and peptides due to the high positronabundance, optimal half-life and short positron range.

Recently, an elegant one step radiolabeling approach was developed forradiofluorination of both small peptides and proteins based on complexbinding of (Al¹⁸F)²⁺ using 1,4,7-triazacyclononane (NOTA) chelator¹⁷⁻²⁰.In this method, the traditional critical azeotropic drying step for18F-fluoride is not necessary, and the labeling can be performed inwater. A number of recently published studies have illustrated thepotential of this new ¹⁸F-labeling method, where successful labeling ofligands for PET imaging of angiogenesis^(21,22), Bombesin²³, EGFR²⁴ andhypoxia²⁵ have been demonstrated.

Various radio-labelled peptide compositions have been developed or areunder development for site-specific targeting of various antigens,receptors and transporters for PET imaging. The general principleinvolves attaching a selected positron emitting radionuclide to apeptide and/or protein having a high specificity for a particularantigen for visualize and quantify the expressing and/or activity levelusing PET imaging. This field of research has shown particularapplicability for tumor diagnosis, staging and treatment monitoring. Aparticularly desirable tumor antigen is uPAR in many different solidtumors including but not limited to non-small cell lung carcinomas,brain tumors, prostate tumors, breast tumors, colorectal tumors,pancreatic tumors and ovarian tumors.

DOTA (1,4,7, 10-tetrakis(carboxymethyl)-1,4,7, 10 tetraazacyclododecane) and its derivatives constitute an important class of chelatorsfor biomedical applications as they accommodate very stably a variety ofdi- and trivalent metal ions. An emerging area is the use of chelatorconjugated bioactive peptides for labelling with radiometals indifferent fields of diagnostic and therapeutic nuclear oncology. NOTAand its derivatives constitute another important class of chelators forbiomedical applications.

uPAR PET imaging has been exploited in several human cancer xenograftmodels using a small linear DOTA-conjugated peptide, DOTA-AE105radiolabeled with ⁶⁴Cu (Li et al, 2008, Persson et al, 2011) and ⁶⁸Ga(Persson et al, 2012) and using NODAGA (NODAGA-AE105) radiolabeled with⁶⁸Ga (Persson et al, 2012).

Malignant tumors are capable of degrading the surrounding extracellularmatrix, resulting in local invasion or metastasis. Urokinase-typeplasminogen activator (uPA) and its cell surface receptor (uPAR) arecentral molecules for cell surface-associated plasminogen activationboth in vitro and in vivo. High expression of uPA and uPAR in many typesof human cancers correlate with malignant tumor growth and associatewith a poor prognosis, possibly indicating a causal role for theuPA/uPAR system in cancer progression and metastasis. Studies byimmunohistochemistry and in situ hybridization indicate that expressionlevels of the components from the uPA system are generally very low innormal tissues and benign lesions. It has also been reported that theuPA/uPAR system is involved in regulating cell-extracellular matrixinteractions by acting as an adhesion receptor for vitronectin and bymodulating integrin function. Based on these properties, the uPA/uPARsystem is consequently considered an attractive target for cancertherapy.

WO 01/25410 describes diagnostically or therapeutically labelleduPAR-targeting proteins and peptides. The peptide or protein comprisesat least 38 amino acid residues, including residues 13-30 of the uPARbinding site of uPA.

U.S. Pat. No. 6,277,818 describes uPAR-targeting cyclic peptidecompounds that may be conjugated with a diagnostic label. The peptidesare based on the amino acid residues 20-30 of uPA.

U.S. Pat. No. 6,514,710 is also directed to cyclic peptides havingaffinity for uPAR. The peptides may carry a detectable label. Thepeptide comprises 11 amino acids joined by a linking unit.

Ploug et al. in Biochemistry 2001, 40, 12457-12168 describes uPARtargeting peptides but not in the context of imaging, including aminoacid sequences as described in the present document. Similar disclosureis provided in U.S. Pat. No. 7,026,282.

The efficient targeting of uPAR demands a selective high-affinity vectorthat is chemically robust and stable.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that [⁶⁸Ga]-, [64Cu]- and[Al¹⁸F]-NOTA-AE105 have superior in vivo characteristics as a uPAR PETligand, with high and specific tumor uptake, thus resulting in a hightumor-to-background ratio and thereby superior contrast as a PET ligandfor uPAR expression tumors. The inventors have found that both [⁶⁸Ga]-and [⁶⁴Cu]-NOTA-AE105 was able to specifically detect uPAR expressinghuman-derived brain tumor lesions in a orthotropic human cancer mousemodel. Moreover, [Al 18F]-NOTA-AE105 was useful to detect uPAR positivehuman prostate cancer lesions after subcutaneously inoculation in mice.Overall, the radiolabeling of NOTA-AE105 with ¹⁸F, ⁶⁸Ga and ⁶⁴Cu, thusenable the visualization and quantification of uPAR using PET Imaging.This is a major improvement in PET Imaging.

The present invention thus provides a positron-emitting radionuclidelabelled peptide conjugate for use in the prediction/diagnosis ofaggressiveness, prognosis, progression or recurrence by PET imaging ofuPAR expressing, and in particular uPAR overexpressed tumors, saidconjugate comprising a uPAR binding peptide coupled via a chelatingagent or covalently to a radionuclide selected from 18F, 64Cu, 68Ga,66Ga, 60Cu, 61Cu, 62Cu, 89Zr, 1241, 76Br, 86Y, and 94mTc, wherein theconjugate is administered in a diagnostically effective amount, such asa dose of 100-500 MBq followed by PET scan ½-24 h after the conjugatehas been administered, and quantification through SUVmax and/or SUVmean.

In a preferred embodiment the peptide is selected from the groupconsisting of:

(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(Ser)-(Leu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser),(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser),(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser),(D-Thr)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([beta]-2-naphthyl-L-alanine)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([beta]-1-naphthyl-L-alanine)-(Ser),(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([beta]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([beta]-1-naphthylL-alanine)-(D-His),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[beta]thoxyethyl)glycine),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile).

For all peptides mentioned, the C-terminal can be either with acarboxylic acid or an amide.

Preferably the chelating agent is DOTA, NOTA, CB-TE2A or NODAGA, andpreferably the peptide is(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)(Trp)-(Ser).

Particularly preferred are the conjugates having the formulas:

The present inventors have surprisingly found that the conjugates of thepresent invention are particularly useful in predicting aggressiveness,prognosis, progression or recurrence by PET imaging of uPAR expressingtumors, in particular prostate, breast, pancreatic, lung, brain andcolorectal cancer.

The present invention also provides a method for predicting/diagnosingthe aggressiveness, prognosis, progression or recurrence of uPARoverexpressed tumors, wherein the method comprises the steps of:

-   -   administrating a conjugate of the present invention in a        diagnostically effective amount, such as a dose of 100-500 MBq;    -   PET scanning ½-24 h after the conjugate has been administered.    -   quantifying through SUVmax and/or SUVmean the absorption/binding        of the conjugate in the tumor.

The steps carried out in accordance with the present invention can besummarized with the flow diagram shown in FIG. 8.

The present conjugates for use in accordance with the present inventioncan discriminate between uPAR expression levels in the primary tumor andmetastases. Also, the use of quantification e.g. SUVmean and especiallySUVmax can predict prognosis, progression and recurrence. Thepositron-emitting radionuclide labelled peptides of the presentinvention specifically target uPAR-positive cancer cells and/or uPARpositive stroma cells surrounding the cancer such as neutrophils andmacrophages, and in particular the most aggressive (metastatic) cells.Moreover, the peptides of the present invention can be used fornon-invasive detection and quantification of the expression level ofuPAR using PET imaging. No current methods measuring uPAR is capable ofthis non-invasively in humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows In vitro competitive inhibition of the uPA:uPAR bindingfor AE105 and AE152 using surface Plasmon resonance.

FIG. 1B shows Radiolabeling method for ¹⁸F-AIF-NOTAAE105.

FIG. 2A shows representative HPLC UV chromatograms of NOTA-AE105.

FIG. 2B shows cold standard AIF-NOTA-AE105.

FIG. 2C shows radio chromatograms for the final product¹⁸F-AIFNOTA-AE105.

FIG. 2D shows radio chromatograms for the final product¹⁸F-AIFNOTA-AE105 and after 30 min in PBS.

FIG. 3A shows Representative PET images after 0.5 h, 1.0 h and 2.0 h p.iof ¹⁸F-AIF-NOTA-AE105 (top) and ¹⁸F-AIF-NOTA-AE105 with a blocking doseof AE152. White arrows indicate tumor.

FIG. 3B shows quantitative ROI analysis with tumor uptake values (%ID/g). A significant higher tumor uptake was found at all three timepoints. Results are shown as % ID/g±SEM (n=4 mice/group). ** p<0.01, ***p<0.001 vs blocking group at same time point.

FIG. 4 shows biodistribution results for ¹⁸F-AIF-NOTA-AE105 (normal) and¹⁸F-AIFNOTA-AE105+blocking dose of AE152 (Blocking) in nude mice bearingPC-3 tumors at 2.5 h p.i. Results are shown as % ID/g±SEM (n=4mice/group). *p<0.05 vs blocking group.

FIG. 5A shows uPAR expression level found using ELISA in PC-3 cells.

FIG. 5B shows uPAR expression level found using ELISA in PC-3 cells inresected PC-3 tumors.

FIG. 5C shows a significant correlation between uPAR expression andtumor uptake was found in the four mice injected with 18F-AIF-NOTA-AE105(p<0.05, r=0.93, n=4 tumors).

FIG. 6 shows in vivo uPAR PET imaging with [⁶⁴Cu]NOTA-A.E105 in aorthotropic human glioblastoma mouse model

FIG. 7 shows in vivo uPAR PET imaging with [⁶⁸Ga]NOTA-AE105 in aorthotropic 5 human glioblastoma mouse model.

FIG. 8 shows a flow diagram summarizing the steps carried out inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, a radiolabeled peptide of the present invention is veryuseful in the prediction of cancer metastasis of uPAR expressing tumors.

The peptides selected for use in the conjugates of the present inventionare typically radiolabeled by coupling a chelating agent to the peptide.The chelating agent is capable of binding a selected radionuclidethereto. The chelating agent and radionuclide is coupled to the peptidein a manner that does not interfere or adversely affect the bindingproperties or specificity of the peptide. The use of various chelatingagents for radio labelling of peptides is well known in the art. Thechelating agent is coupled to the peptide by standard methodology knownin the field of the invention and may be added at any location on thepeptide provided that the biological activity of the peptide is notadversely affected. Preferably, the chelating group is covalentlycoupled to the amino terminal amino acid of the peptide. The chelatinggroup may advantageously be attached to the peptide during solid phasepeptide synthesis or added by solution phase chemistry after the peptidehas been obtained. Preferred chelating groups include DOTA, NOTA, NODAGAor CB-TE2A.

Concerning the synthesis of the peptides used in the present inventionreference is made to U.S. Pat. No. 7,026,282.

The peptide/chelate conjugates of the invention are labeled by reactingthe conjugate with radionuclide, e.g. as a metal salt, preferably watersoluble. The reaction is carried out by known methods in the art.

The conjugates of the present invention are prepared to provide aradioactive dose of 35 between about 100-500 MBq (in humans), preferablyabout 200-400 MBq, to the individual. As used herein, “a diagnosticallyeffective amount” means an amount of the conjugate sufficient to permitits detection by PET. The conjugates may be administered intravenouslyin any conventional medium for intravenous injection. Imaging of thebiological site may be effected within about 30-60 minutespost-injection, but may also take place several hours post-injection.Any conventional method of imaging for diagnostic purposes may beutilized.

The following example focuses on the specific conjugate denoted¹⁸F-AIF-NOTA-AE105. Other conjugates within the scope of the claimsherein will be apparent to one skilled in the art from consideration ofthe specification or practice of the invention as disclosed herein.

The following chemistry applies to the Examples:

AE105 (Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser—OH) (1)

The peptide according to the above mentioned sequence was synthesized bystandard solid-phase peptide chemistry.

NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser—OH)

The product is purified by RP-HPLC and analysed by RP-HPLC (retensiontime: 11.5 min, purity >98%) and electrospray-MS (1510.8 m.u.).

Example 1

The aim of the present study was to synthesize a NOTA-conjugated peptideand use the Al¹⁸F method for development for the first ¹⁸F-labeled PETligand for uPAR PET imaging and to perform a biological evaluation inhuman prostate cancer xenograft tumors. To achieve this, the presentinventors synthesized high-affinity uPAR binding peptide denoted AE105and conjugated NOTA in the N-terminal. ¹⁸F-labeling was done accordingto a recently optimized protocol²⁶. The final product(¹⁸F-AIF-NOTA-AE105) was finally evaluated in vivo using both microPETimaging in human prostate tumor bearing animals and after collection oforgans for biodistribution study.

Chemical Reagents

All chemicals obtained commercially were of analytical grade and usedwithout further purification. No-carrier-added ¹⁸F-fluoride was obtainedfrom an in-house PETtrace cyclotron (GE Healthcare). Reverse-phaseextraction C18 Sep-Pak cartridges were obtained from Waters (Milford,Mass., USA) and were pretreated with ethanol and water before use. Thesyringe filter and polyethersulfone membranes (pore size 0.22 μm,diameter 13 mm) were obtained from Nalge Nunc International (Rochester,N.Y., USA). The reverse-phase HPLC using a Vydac protein and peptidecolumn (218TP510; 5 μm, 250×10 mm) was performed as previouslydescribed²¹.

MicroPET scans were performed on a microPET R4 rodent model scanner(Siemens Medical Solutions USA, Inc., Knoxville, Tenn., USA). Thescanner has a computer-controlled bed and 10.8-cm transaxial and 8-cmaxial fields of view (FOVs). It has no septa and operates exclusively inthe three-dimensional (3-D) list mode. Animals were placed near thecenter of the FOV of the scanner.

Peptide Synthesis, Conjugation and Radiolabeling

NOTA-conjugated AE105(NOTA-Asp-Cha-Phe-(D)Ser-(D)Arg-Tyr-Leu-Trp-Ser-COOH) was purchased fromABX GmbH. The purity was characterized using HPLC analysis and the masswas confirmed using matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF-MS) (Se Suppl. FIG. 1A). Theradiolabeling of NOTAAE105 with ¹⁸F-AIF is shown in FIG. 1 and was doneaccording to a recently published protocol with minor modifications²⁶.

In brief, a QMA Sep-Pak Light cartridge (Waters, Milford, Ma, USA) wasfixed with approximately 3 GBq of 18F-fluoride and then washed with 2.5ml of metal free water. Na18F was then eluted from the cartridge with 1ml saline, from which 100 μl fraction was taken. Then amounts of 50μ10.1 M Na-Acetate buffer (pH=4), 3 μl 0.1 M AlCl₃ and 100 μl of Na¹⁸F in0.9% saline (300 MBq) were first reacted in a 1 ml centrifuge tube(sealed) at 100° c. for 15 min. The reaction mixture was cooled. 50μ1ethanol and 30 nmol NOTA-AE105 in 3 μl DMSO were added and the reactionmixture were heated to 95° C. for 5 min. The crude mixture was purifiedwith a semi-preparative HPLC. The fractions containing¹⁸F-AIF-NOTA-AE105 were collected and combined in a sterile vial. Theproduct was diluted in phosphate-buffered saline (PBS, pH=7.4) so anyorganic solvents were below 5% (v/v) and used for in vivo studies.

Cell Line and Animal Model

Human prostate cancer cell line PC-3 was obtained from the American TypeCulture Collection (Manassas, Va., USA) and culture media DMEM wasobtained from Invitrogen Co. (Carlsbad, Calif., USA). The cell line wascultured in DMEM supplemented with 10% (v/v) fetal bovine serum and 1%(v/v) penicillin/Streptomycin at 37° C. and 5% CO₂. Xenografts of humanPC-3 prostate cancer cells were established by injection of 200 μl cells(1×10⁸ cells/ml) suspended in 100 μl Matrigel (BO Biosciences, San Jose,Calif., USA), subcutaneously in the right flank of male nude miceobtained from Charles River Laboratory (Wilmington, Mass. USA), Tumorswere allowed to grow to a size of 200-500 mg (3-4 weeks).

MicroPET Imaging

Three min static PET scans were acquired 0.5, 1.0 and 2.0 h postinjection (p.i) of ¹⁸FAIF-NOTA-AE105 via tail-vein injection of 2-3 MBq(n=4). Similar, the blocking study was performed by injection of theligand together with 100 μg of AE152 (uPAR antagonist) through the tailvein (n=4) and PET scanned at the same time points. During each threeminutes PET scan, mice were anesthetized with isoflurane (5% inductionand 2% 30 maintenance in 100% 02). Images were reconstructed using atwo-dimensional ordered subsets expectation maximization (OSEM-2D)algorithm. No background correction was performed. All results wereanalyzed using Inveon software (Siemens Medical Solutions) and PET datawas expressed as percent of injected dose per gram tissue (% ID/g) basedon manual region-of-interest drawing on PET images and the use of acalibration constant. An assumption of a tissue density of 1 g/ml wasused. No attenuation correction was performed.

Biodistribution Studies

After the last PET scan, all PC-3 bearing mice were euthanized. Blood,tumor and major organs were collected (wet-weight) and the radioactivitywas measured using a y-counter from Perkin Elmer, Mass., USA (N=4mice/group).

uPAR ELISA

uPAR ELISA on resected PC-3 tumors was done as described previously indetail¹⁵. All results were performed as duplicate measurements.

Statistical Analysis

All quantitative data are expressed as mean±SEM (standard error of themean) and means were compared using Student t-test. Correlationstatistics was done using linear regression analysis. A P-value of s0.05 were considered statistically significant.

uPAR Binding Affinity

The uPAR binding affinity of AE105 and AE152²⁷ (used for blockingstudies) was in this 20 study found to be 14.1 nM and 2.9 nM,respectively (FIG. 1A). A high uPAR binding affinity for AE105 withdifferent chelators conjugated in the N-terminal, including the NOTAanalogue NODAGA, has been confirmed in our previously studies^(15, 16),thus confirming the ability to make modifications in the N-terminal ofAE105 without losing affinity towards human uPAR^(14, 27, 28).

Radiochemistry

The ¹⁸F-labeling of NOTA-AE105 was synthesized based on a recentlypublished procedure with some modification (FIG. 1B). During ourlabeling optimization, we found that 33% ethanol (v/v) was optimal using30 nmol NOTA-AE105. We first formed the 18F-AIF complex in buffer at100° C. for 15 min. Secondly was the NOTA-conjugated peptide added andincubated together with Ethanol at 95° C. for 5 min. By adding ethanolwe were able to increase the overall yield to above 92.7% (FIG. 2C),whereas the yield without ethanol was only 30.4%, with otherwise sameconditions. No further increase in the overall yield was observed usinglonger incubation time and/or different ethanol concentrations or usingless than 30 nmol conjugated peptide. Two isomers were observed for¹⁸F-AIF-NOTA-AE105.

In order to ensure the formation of the right product, a cold standardof the final product was synthesis (AIF-NOTA-AE105). The HPLC analysisof the precursor (NOTA-AE105, FIG. 2A) confirmed the purity of theNOTA-conjugated precursor (>97%) and MALDI-MS confirmed the mass (1511.7Da) (See suppl. FIG. 1). The cold standard (AIFNOTA-AE105, FIG. 2B),with the right mass confirmed by MALDI-MS (1573.6 Da) (See suppl. FIG.1B), corresponded well in regards to retention time with the ‘hot’product (FIG. 2C), thus confirming the formation of 18F-AIF-NOTA-AE105(FIG. 2C). No degradation of the final product was found after 30 min inPBS (FIG. 2D). The radioactive peaks were collected and diluted in PBSand used for in vivo studies. The specific activity in the final productwas above 25 GBq/μmol.

In Vivo PET Imaging

¹⁸F-AIF-NOTA-AE105 was injected i.v. in four mice bearing PC-3 tumorsand PET scan were performed 0.5, 1.0 and 2.0 h post injection (p.i).Tumor lesions were easily identified from the reconstructed PET images(FIG. 3A) and ROI analysis revealed a high tumor uptake, with 5.90±0.35%ID/g after 0.5 h, declining to 4.22±0.13% ID/g and 2.54±0.24% ID/g after1.0 and 2.0 h, respectively (FIG. 3B).

In order to ensure that the found tumor uptake did indeed reflectspecific uPAR mediated uptake, four new PC-3 tumor bearing mice werethen injected with a mixed solution containing ¹⁸F-AIF-NOTA-AE105 and100 μg of the high-affinitty uPAR binding peptide denoted AE152, inorder to see if the tumor uptake could be inhibited. A significant loweramount of ¹⁸F-AIF-NOTA-AE105 tumor uptake was found at all three timepoints investigate (FIG. 3B) and tumor lesions were not as easilyidentified in the PET images (FIG. 3A). At 1.0 h p.i a tumor uptake of1.86±0.14% ID/g was found in the blocking group compared with 4.22±0.13%ID/g found in the group of mice receiving only ¹⁸F-AIFNOTA-AE105(p<0.001, 2.3 fold reduction).

Biodistribution

After the last PET scan, each group of mice where euthanized andselected organs and tissues were collected to investigate thebiodistribution profile 2.5 h p.i. (FIG. 4). A significant higher tumoruptake in the group of mice receiving ¹⁸F-AIF-NOTA-AE105 was foundcompared with blocking group (1.02±0.37% ID/g vs. 0.30±0.06% ID/g,p<0.05), thus confirming the specificity of ¹⁸F-AIF-NOTA-AE105 for humanuPAR found in the PET study. Highest activity was found in the kidneysfor both groups of mice, confirming the kidneys to be the primarilyroute of excretion. Beside kidneys, the bone, well known to accumulatefluoride, also had a relatively high uptake of 3.54±0.32% ID/g and2.34±0.33% ID/g for normal and blocking group, respectively.

uPAR Expression

Both the PC-3 cells used for tumor inoculation and all PC-3 tumors atthe end of the study (n=8) were finally analyzed for confirmingexpression of human uPAR (FIG. 5). An expression in the cells of6.53±1.6 ng/mg protein was found (FIG. 5A), whereas the expression levelin the resected tumors was 302±129 pg/mg tumor tissue (FIG. 5B). Asignificant correlation between tumor uptake of 18F-AIF-NOTA-AE105 anduPAR expression was found (p<0.05, r=0.93) (FIG. 5C).

Data Interpretation

The above experiments provide evidence for the applicability of an18F-labeled ligand for 15 uPAR PET. The ligand was characterized in ahuman prostate cancer xenograft mouse model. Based on the obtainedresults, similar tumor uptake, specificity and tumor-to-backgroundcontrast were found compared to our previously published studies using64Cu- and 68Ga-based ligands for PET^(15, 16). Based on the superiorphysical characteristics of 18F and the high tumor-to-backgroundcontrast found already after 1 h p.i, our new 18F-based ligand must beconsidered the so far most promising uPAR PET candidate for translationinto clinical use in order to non-invasively characterize invasivepotential of e.g. prostate cancer.

¹⁸F-labeling of peptides using the AIF-approach has previously beendescribed to be performed at 100° c. for 15 min, at pH=4¹⁷⁻²⁰. Thisprotocol was modified, since degradation of the NOTA-conjugated peptidewas observed using these conditions. The present inventors thereforefirst produced the ¹⁸F-AIF complex using the above mentioned conditionsand next added the NOTA-conjugated peptide and lowered the temperatureto 95° C., and within 5 min obtained a labeling yield of 92.7% and withno degradation of the peptide. Two isomers of ¹⁸F-AIF-NOTA-AE105 wereproduced. Same observations have been reported by others for¹⁸F-AIF-NOTA-Octreotide¹⁸ and all NOTA-conjugated IMP peptide analoguesdescribed¹⁹. The ratio of the two peaks were nearly constant for eachlabeling and both radioactive peaks were collected and used for furtherin vivo studies. This approach was recently also described by others²⁶.

Besides optimizing the temperature and time, the present inventors foundthat the addition of ethanol, to a final concentration of 33% (v/v),resulted in a significant higher labeling yield, compared withradiolabeling without ethanol (30.4% vs 92.7%), using the same amount ofNOTA-conjugated peptide. Same observations have recently been describedby others²⁶. Here the effect of lowering the ionic strength wasinvestigated using both acetonitrile, ethanol, dimethylforamide (DMF)and tetrahydrofuran (THF) at different concentrations. A labeling yieldof 97% was reported using ethanol at a concentration of 80% (v/v).However, they used between 76-383 nmol NOTA-conjugated peptide, whereasin this study only used 30 nmol was used. The amount needed for optimallabeling yield therefore seems to be dependent on the peptide and on theamount of peptide used for labeling.

The tumor uptake of ¹⁸F-AIF-NOTA-AE105 was similarly compared withpreviously published results pertaining to ⁶⁴Cu-based ligands¹⁵. Thetumor uptake 1 h p.i was 4.79±0.7% ID/g, 3.48±0.8% ID/g and 4.75±0.9%ID/g for ⁶⁴Cu-DOTA-AE105, ⁶⁴Cu-CB-TE2A-AE105, ⁶⁴Cu-CB-TE2A-PA-AE105compared to 4.22±0.1% ID/g for ¹⁸F-AIF-NOTA-AE105. However, all⁶⁴Cu-based ligands were investigated using the human glioblastoma cellline U87MG, whereas in this study, the prostate cancer cell line PC-3was used. Considering that the data show that the level of uPAR in thetwo tumor types is not similar, with PC-3 having around 300 pg uPAR/mgtumor tissue (FIG. 5B) and U87MG having approximately 1,700 pg/mg tumortissue (unpublished), the tumor uptake of ¹⁸F-AIF-NOTA-AE105 seems to berelatively higher per pg uPAR, However, a direct comparison between thetwo independent studies is difficult, considering the different cancercell line used. However, the present inventors have previously shown asignificant correlation between uPAR expression and tumor uptake acrossthree tumor types¹⁵, which is confirmed in the present study using PC-3xenografts (FIG. 5C), further validating the ability of¹⁸F-AIF-NOTA-AE105 to quantify uPAR expression using PET imaging. TheuPAR specific binding of ¹⁸F-AIF-NOTA-AE105 in the present study wasconfirmed by a 2.3-fold reduction in tumor uptake of ¹⁸F-AIF-NOTA-AE1051 h p.i. when co-administration of an uPAR antagonist (AE152) wasperformed for blocking study.

The biodistribution study of 18F-AIF-NOTA-AE105 confirmed the kidneys tobe the primary route of excretion and the organ with highest level ofactivity (FIG. 4). Same excretion profiles have been found for⁶⁸Ga-DOTA/NODAGA-AE105¹⁶, ¹⁷⁷Lu-DOTAAE105³⁰. Besides the kidneys andtumor, the bone also had a relatively high accumulation of activity.Bone uptake following injection of 18F-based ligands is a well-describedphenomenon and used clinically in NaF bone scans³¹. A bone uptake of3.54% ID/g 2.5 h p.i was found, which is similar to the bone uptakefollowing ¹⁸F-FDG injection in mice, where 2.49% ID/g have been reported1.5 h p.i.¹⁷.

The development of the first ¹⁸F-based ligand for uPAR PET provides ofnumber of advantages compared to previously published ⁶⁴Cu-based uPARPET ligands. Considering the optimal tumor-to-background contrast asearly as 1 h p.i. as found in this study and in previously studies using64Cu, the relatively shorter half-life of ¹⁸F (T_(1/2)=1.83 h) comparedwith ⁶⁴Cu (T_(1/2)=12.7 h) seems to be optimal consider the much lowerradiation burden to future patients using ¹⁸F-AIF-NOTA-AE105. Moreover,is the production of ¹⁸F well established in a number of institutionsworldwide, whereas the production of ⁶⁴Cu still is limited to relativelyfew places.

Example 2 [⁶⁴Cu]NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH)

⁶⁴CuCl2 dissolved in 50 ul metal-free water was added to a solutioncontaining 10 nmol NOTA-AE105 and 2.5 mg gentisic acid dissolved in 500ul 0.1 M NH4OAc buffer (pH 5.5) and left at room temperature for 10minutes resulting in 375 MBq [64Cu]NOTA-AE105 20 with a radiochemicalpurity above 99%. The radiochemical purity decreased to 94% after 48hours storage.

Example 3 In Vivo uPAR PET Imaging with [⁶⁴Cu]NOTA-AE105 in aOrthotropic Human Glioblastoma Mouse Model

A mouse was inoculated with human derived glioblastoma cells in thebrain. 3 weeks later a small tumor was visible using microCT scan AmicroPET images was recorded 1 hr post i.v. injection of approximately 5MBq [⁶⁴Cu]NOTA-AE105. Uptake in the tumor and background brain tissuewas quantified. Moreover, was a control mouse (with no tumor inoculated)also PET scanned using the same procedure, to investigate the uptake innormal brain tissue with intact blood brain barrier. See FIG. 6.

Example 4 [68Ga]NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH)

A 1 ml fraction of the eluate form a ⁶⁸Ge/68Ga generator for added to asolution containing 20 nmol NOTA-AE105 dissolved in 1000 ul 0.7M NaOAcbuffer (pH 3.75) and heated to 60° C. for 10 minutes. The correspondingmixture could be purified on a C18 SepPak column resulting in 534MBq[⁶⁸Ga]NOTA-AE105 with a radiochemical purity above 98%.

Example 5 In Vivo uPAR PET Imaging with [⁶⁸Ga]NOTA-AE105 in aOrthotropic Human Glioblastoma Mouse Model

A mouse was inoculated with human derived glioblastoma cells in thebrain. 3 weeks 15 later a small tumor was visible using microCT scan AmicroPET images was recorded 1 hr post i.v. injection of approximately 5MBq [68Ga]NOTA-AE105. Uptake in the tumor and background brain tissuewas quantified. Moreover, was a control mouse (with no tumor inoculated)also PET scanned using the same procedure, to investigate the uptake innormal brain tissue with intact blood brain barrier. See FIG. 7.

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1. A method of generating images of uPAR expression in a human bydiagnostic imaging of uPAR expressing tumors involving administering animaging agent to said human, and generating an image of at least a partof said body to which said imaging agent is administered; wherein theimaging agent is a positron-emitting radionuclide labelled peptideconjugate, said conjugate comprising a uPAR binding peptide coupled viathe chelating agent DOTA to a ⁶⁴Cu radionuclide; wherein the conjugateis to be administered in a dose of 100-500 MBq followed by PET scanning½-24 h after the conjugate has been administered, and quantificationthrough SUVmax and/or SUVmean; wherein the peptide is selected from thegroup consisting of:(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(Ser)-(Leu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser),(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser),(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser),(D-Thr)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([beta]-2-naphthyl-L-alanine)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([beta]-1-naphthyl-L-alanine)-(Ser),(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([beta]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([beta]-1-naphthyl-L-alanine)-(D-His),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[beta]thoxyethyl)glycine),(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile),wherein the C-terminal is either a carboxylic acid or an amide.


2. The method according to claim 1, wherein the peptide is(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser).3. The method according to claim 1, having the formula:


4. The method according to claim 1, wherein the cancer is selected fromprostate, breast, pancreatic, lung, brain and colorectal cancer.
 5. Themethod according to claim 1, wherein the conjugate is administered in adose of 200-400 MBq.
 6. The method according to claim 1, wherein theimaging agent is provided in a pharmaceutical composition comprising theimaging agent, together with one or more pharmaceutical acceptableadjuvants, excipients or diluents.